Abstract
The lack of effective disease-modifying therapies for axonopathies highlights the need for novel preclinical models suitable for treatment development. Two-dimensional neuronal cultures lack the directional axonal distribution required to investigate length-dependent processes such as peripheral neuropathies. To optimize this well-established model system, we developed a robust human platform to study axonal morphology and physiology based on three-dimensional motor neuron cultures (i.e., spinal spheroids). We differentiate motor neurons from human induced pluripotent stem cells, purify them by magnetic sorting, and culture them in suspension until they form spheroids. Axons are allowed to grow out of plated spinal spheroids in a radial fashion at an average rate of 200 micrometers/day and reach up to 1 cm in length. This system is optimized for morphological analysis, including high content imaging, investigation of axonal protein expression, and time-lapse imaging of axonal transport.